This research program investigates immune responses induced by viral and plasmid vectors. Results are relevant to vaccine potency and mechanisms of action. In addition, immune responses can alter the safety and efficacy of gene therapy using viral or plasmid vectors, especially in the context of readministration, either by blocking therapy or by causing immunopathology. Better understanding of such immune responses can help select predictors of clinical success as well as adverse events, and thus contribute to improved regulatory decision-making. Our work focuses on the mouse influenza system. Main projects: a) Heterosubtypic immunity: Animals immunized against influenza A virus have cross-protection against challenge with flu A of different subtypes. Vaccine development and prevention of pandemics would be aided by a more complete understanding of the broad cross-protection against widely divergent viral strains that is observed in animals. Ongoing studies examine the roles of antibodies and of CD4+ and CD8+ T cells in heterosubtypic immunity, as well as its specificity. b) Immune responses induced by plasmid DNA: DNA vaccines encoding internal influenza virus antigens reduce challenge virus replication, and resulting morbidity and mortality. Other investigators had reported that classical CD8+ CTL were required for protection by influenza DNA vaccination. Surprisingly, our results show that depletion of CD8+ T cells (verified by flow cytometry and CTL assays on cells from the infected lungs) had no significant effect on survival of challenge infection. This indicates that either CD4+ or CD8+ T cells can function without the other subset to protect the animal. We are currently investigating mechanisms by which CD4+ cells protect in the absence of CD8+ cells. c) One advantage of such DNA vaccination would be the broad range of viral variants against which it might protect. We are currently studying the effect of DNA vaccines encoding internal antigens of influenza virus A/PR/8 (H1N1) on subsequent challenge with other subtypes. d) Many vaccines based on T cell immunity (HIV, hepatitis, flu) have been proposed when antibodies are ineffective or too narrow in viral strain specificity, but the protective efficacy of T cells acting in the absence of antibody has not been tested in most cases. Using mice lacking all antibodies due to disruption of Ig H and L chain genes, we have previously shown protection against influenza B challenge, and have demonstrated that optimal protection requires both CD4+ and CD8+ T cells. We have now extended those studies to the influenza A virus system. e) The role of local, mucosal immune responses in cross-protection is not well understood. We are performing mucosal immunizations and challenges in various mouse strains, to analyze the role of mucosal immunity in cross-protection of the upper and lower respiratory tract.